TECHNICAL FIELD OF THE INVENTION
The present invention relates to a dispensing amount control method for dispensing with high accuracy while confirming the dispensing amount, particularly when dispensing a small amount of sample or sample.
A conventional method of controlling the dispensed volume is generally a method of controlling the volume change of the syringe, and the dispensed volume is almost tens of μL or more in most cases. As a method for detecting such a dispensed amount, there has been proposed a method of measuring the dispensed amount from a projected image of a sample being discharged using an optical system (for example, see Patent Documents 1 and 2). .
Further, as a method of measuring the volume of a sample of about 10 μL, which is the size to which water is dropped, a method of measuring the volume from a projected image using a light receiving element group such as a CCD (charge coupled device) has been proposed. (See, for example, Patent Document 3).
As described in Patent Document 2, a method of determining whether or not the dispensed amount is normal using the above-described volume measurement device has been proposed.
Also, recently, the demand for dispensing accuracy is high with the minimization of dispensing volume, and a method of applying a small amount of droplets by applying inkjet technology has been proposed. There is a problem that it is not injected.
[Patent Document 1]
JP-A-5-2025 [Patent Document 2]
JP-A-5-223830 [Patent Document 3]
[Problems to be solved by the invention]
In the above-described prior art, when dispensing (dispensing) a desired dispensing amount, it is not certain that the desired dispensing amount has been dispensed simply by controlling the volume change of the syringe, and confirmation is not possible. There is a problem that can not be. In addition, a method of measuring the dispensed volume using a volume measuring device and determining whether the dispensed volume is normal is a method of determining the result, and the dispensed volume itself is controlled with high precision. It does not do.
An object of the present invention is a dispensing amount control method capable of dispensing a desired dispensing amount with high accuracy while measuring the dispensing amount, particularly in a dispensing operation in which the dispensing amount is several tens μL or less. Is to provide.
[Means for Solving the Problems]
The above-described object is a dispensing head that converts a small amount of sample into droplets from a pipette having a hole at the tip and dispenses the droplet, a droplet volume sensor that detects the volume of the droplet flying from the dispensing head in a non-contact manner, In a dispensing apparatus including a controller that controls a drive of a dispensing head and calculates a volume of a droplet from an output signal of a droplet volume sensor, the dispensing head uses the dispensing head to remove a liquid from a pipette hole. Achieved by a step of discharging a droplet, a step of calculating a volume of a droplet in flight by a droplet volume sensor, and a step of controlling a desired dispensed amount by feeding back volume information obtained in the step. Is done.
BEST MODE FOR CARRYING OUT THE INVENTION
An embodiment of the present invention will be described in detail with reference to the drawings. FIG. 1 is a dispensing apparatus 10 for realizing the dispensing amount control method of the present invention, and is a partial cross-sectional view of the dispensing apparatus 10 as viewed from a side. The configuration of the dispensing device 10 shown in FIG. 1 will be described below. The pipette 1 having a hole with a diameter of about 500 μm at the tip is fixed by a pipette grip 6 with a screw 6 a. The pipette holding part 6 may fix the pipette 1 by a press-fitting method. The piston 2 which advances and retreats in contact with the inner wall surface of the pipette 1 is fixed by a screw 5 a by a piston gripper 5. The piston gripper 5 may fix the piston 2 by a press-fitting method, or may grip the piston 2 head by a chuck mechanism. One end of the piezoelectric actuator 11 is bonded to the piston gripper 5, and the other end of the piezoelectric actuator 11 is bonded to the shaft 4 of the linear stepping motor 3. As a method for fixing the piezoelectric actuator 11, a screw or the like may be used. The shaft 4 of the linear stepping motor 3 linearly moves, and the piston 2 and the piezoelectric actuator 11 move up and down by the linearly moving shaft 4. The linear stepping motor 3, the pipette grip 6 and the hinge 13 are fixed to the base 7. A droplet volume sensor 12 that detects the volume of the droplet 8a in a non-contact manner is fixed to the hinge 13, and the ring-shaped hollow droplet volume sensor 12 is disposed below the hole of the pipette 1.
The controller 20 of the dispensing device 10 configured as described above includes a step motor driver 22, a piezoelectric driver 23, a volume calculation unit 24, and a control unit 21 for controlling these. By transmitting a pulse train signal from the control unit 21 to the step motor driver 22, the speed and position of the piston 2 can be controlled in the forward and backward directions. The piezoelectric driver 23 is, for example, a high-speed amplifier that amplifies an analog voltage signal from 0 V to 5 V DC output from the control unit 21 from 0 V to 150 V DC. Displace. This minute displacement amount is, for example, about 50 μm, and the piston 2 is displaced at high speed to apply pressure to the sample 8 and to push it out of the hole. As a result, about tens of nL from the hole is obtained. The droplet 8a is ejected. When the droplet 8 a is injected into the container 9 through the hollow droplet volume sensor 12, the volume of the droplet 8 a is calculated and measured from the output signal of the droplet volume sensor 12 by the volume calculator 24.
Hereinafter, a method for measuring the volume of the droplet 8a will be described with reference to FIGS. 4, 5, 6, and 7. FIG. FIG. 4 shows a block circuit of the droplet volume sensor 12 and the volume calculation unit 24. The droplet volume sensor 12 includes two pairs of light emitting and light receiving elements. The light emitted from the first light emitting element 30a is formed so as to hit only the first light receiving element 30b, and similarly, the light emitted from the second light emitting element 31a hits only the second light receiving element 31b. ing. Two pairs of light-emitting and light-receiving elements are arranged so that the two lights are located at positions separated by a distance h in the direction in which the droplet 8a passes.
The light emitting side and the light receiving side will be described with reference to FIG. 5 showing a pair of light emitting and light receiving elements. The light emitting elements 30a and 31a convert light emitted from a device 40 such as a light emitting LED or a semiconductor laser into a parallel light beam by a lens 41, and form an extremely long light beam by a shielding plate. Each of the light receiving elements 30b and 31b is a photodiode having a shielding plate 43 or the like, and receives a projected image of the droplet 8a. FIG. 6 is a diagram showing the light beam 45 and the droplet 8a as viewed from the light receiving side. As shown in FIG. 6, the formed light beam 45 is thin in the vertical direction and long in the horizontal direction. The reason for this is that the droplet 8a is elongated because it captures an extremely fine horizontal cross-sectional image of the droplet 8a and because the droplet 8a does not necessarily pass through the center of the light beam 45.
Next, a block circuit diagram of the volume calculation unit 24 shown in FIG. 4 will be described. The voltage signal of the cross-sectional image of the droplet 8a output from the droplet volume sensor 12 is amplified through amplifiers 32 and 33, converted from analog signals into digital values by A / D converters 34 and 35, and stored in memories 36 and 37. To memorize. A predetermined frequency f, for example, 10 MHz, is output from the clock 39 at the sampling period, and at the same time, the address counters (not shown) of the memories 36 and 37 are incremented, and the projected images of the droplets 8a are sequentially stored in the memories 36 and 37. Go. The timing for storing the above-described projected image of the droplet 8a is when the piezoelectric driver 23 is driven, and receives a signal (not shown) from the upper control unit 21. At this time, the CPU 38 provided with the ROM 38a in which a program is stored in advance outputs a Lo level signal to a Hi level signal from the port P1 terminal. The frequency oscillated from the clock 39 is output from the three-state output element 39a, and starts the operation of storing the projected images in the memories 36 and 37. When a predetermined time passes when the droplet 8a passes through the droplet volume sensor 12, the CPU 38 outputs a Lo level signal from the port P1 terminal, and ends the operation of storing the projection image.
When the operation of storing the projection image is completed, data as shown in FIG. 7 is stored in the memories 36 and 37, and the CPU 38 calculates the volume of the droplet 8a from these data. First, the flight speed v of the droplet 8a is determined. The flight speed v is calculated from the distance h between two rays and the transit time t therefrom.
v = h / t
It becomes. Since the sampling period (1 / f) seconds is a fixed period, the passing time t is obtained from the interval at which the projection image data of the two memories 36 and 37 starts to change. When the volume during one sampling from the projection images of the droplets 8a regarded as fine columnar V i, since it is the product of cross-sectional area and height,
Height of micro cylinder = v (1 / f)
Cross-sectional area of micro cylinder = π (radius) 2 = π (y i · α) 2
V i = π (y i · α) 2 × v (1 / f)
Here, y i is the amount of shadow projection image data, which is regarded as the radius of the small cylinder V i by multiplying the correction coefficient alpha. The correction coefficient α can be obtained experimentally by projecting a known object such as a cylinder.
The volume V of the droplet 8a is 1 seeking small cylinder V i during the sampling, it is obtained by sum of products of all the data number n pieces recessed portion of the projection image data (Equation 1).
As a method of increasing the calculation accuracy of the volume measurement of the droplet 8a, the volumes may be calculated and averaged from the projection image data stored in the two memories 36 and 37, respectively. The two light beams of the droplet volume sensor 12 may be parallel. However, it is preferable to project the light beams orthogonally from different angles, calculate the respective volumes, and average the volumes.
This will be described below with reference to the flowcharts shown in FIGS. First, the step of sucking the sample 8 into the pipette 1 using the above-described dispensing apparatus 10 will be described with reference to FIG.
At the start of suction in step 200, the shaft 4 of the linear stepping motor 3 is driven to lower the piston 2 to the lowermost end. Next, the droplet volume sensor 12 is pulled by a wire or the like (not shown), and a sensor retreating step 210 for opening a space below the pipette 1 is executed. Subsequently, a step 220 of immersing the tip of the pipette 1 in the parent sample 8 and driving the linear stepping motor 3 to pull up the piston 2 and aspirate a predetermined dispensed amount into the pipette 1 is executed. After the suction is completed, after the tip of the pipette 1 is separated from the parent sample 8, a step 230 of setting the retracted droplet volume sensor 12 below the hole of the pipette 1 is executed. Thus, the suction operation ends (Step 240).
Next, a method of moving the dispensing device 10 above the container 9 to be dispensed and controlling the dispensed amount while measuring the volume of the droplet 8a will be described with reference to FIG.
When the start of dispensing (step 250) is instructed, the control unit 21 minutely displaces the piezoelectric actuator 11 at a high speed to cause the droplet 8a to fly from the hole at the tip of the pipette 1 (step 260). The volume of the flying droplet 8a is obtained by the volume calculation unit 24 in the manner described above (step 270). Since the liquid surface of the hole at the tip of the pipette 1 enters the pipette 1 by the amount of the discharged droplet 8a, the linear stepping motor 3 is driven to feed the piston 2 in step 280. Since the inner diameter of the pipette 1 is known, the movement amount is obtained from the volume of the discharged droplet 8a. In step 290, the product sum of the discharged droplets 8a is obtained. It is determined whether or not the discharged dispensed amount, that is, the sum-of-products amount is a desired dispensed amount (step 300). When the desired dispensing amount is reached, the dispensing operation ends (step 310).
Further, the controller 20 can store the dispensed amount of each individual, and can communicate the dispensed amount to a host computer or the like, and can perform data management of the dispensed amount of a valuable sample or the like.
【The invention's effect】
According to the present invention, there is provided a dispensing head for dispensing a small amount of sample from a pipette having a hole at the tip into droplets, and a droplet volume sensor for detecting the volume of a droplet flying from the dispensing head in a non-contact manner. In a dispensing apparatus having a controller having a volume operation unit for controlling the drive of a dispensing head and calculating the volume of a droplet from an output signal of a droplet volume sensor, the dispensing head uses A step of discharging a droplet, a step of calculating the volume of the droplet in flight with a droplet volume sensor, and a step of controlling a desired dispensing amount by feeding back the volume information obtained in this step, A highly accurate and reliable dispensing amount control method can be provided.
[Brief description of the drawings]
FIG. 1 is a partial cross-sectional view of a dispensing apparatus of one embodiment according to a dispensing amount control method of the present invention as viewed from a side.
FIG. 2 is a flowchart illustrating an embodiment of a dispensing amount control method according to the present invention.
FIG. 3 is a flowchart illustrating an embodiment of a dispensing amount control method according to the present invention.
FIG. 4 is a block circuit diagram of a volume calculation unit of one embodiment according to the dispensing amount control method of the present invention.
FIG. 5 is a side view illustrating an optical system of a droplet volume sensor according to an embodiment of the present invention, which relates to a dispensing amount control method.
FIG. 6 is a cross-sectional view illustrating a light beam of the droplet volume sensor according to one embodiment of the dispensing amount control method of the present invention.
FIG. 7 is an image diagram in which a projected image of a droplet is stored in a memory according to one embodiment of the dispensing amount control method of the present invention.
[Explanation of symbols]
1 is a pipette, 2 is a piston, 3 is a linear stepping motor, 4 is a shaft, 8 is a sample, 8a is a droplet, 10 is a dispensing device, 11 is a piezoelectric actuator, 12 is a droplet volume sensor, 20 is a controller, 24 Is a volume calculation unit, 260 is a step of discharging a droplet, 270 is a step of calculating the volume of a droplet, and 300 is a step of controlling a desired dispensed amount by feeding back volume information.